Human erythropoietin gene; high level expression in stably transfected mammalian cells

ABSTRACT

The Apa I restriction fragment of the human erythropoietin gene, for producing high titers of biologically active hormone from stably transfected cell lines.

This invention was made in part with Government support under ResearchGrants HL 16919, AM 19410, AM 01418, and CA 31615 awarded by theNational Institutes of Health. The Government has certain rights in thisinvention.

This application is a continuation of U.S. application Ser. No.07/453,381, filed Dec. 18, 1989, now abandoned which is a continuationof Ser. No. 07/211,278, filed Jun. 21, 1988, now abandoned, which is aContinuation of Ser. No. 06/820,423, filed Jun. 22, 1986, now abandoned.

FIELD OF THE INVENTION

This invention relates generally to the field of genetic engineering,particularly to the expression of glycoprotein products of recombinantgenes, and more particularly to the expression of high levels ofbiologically active human erythropoietin from stably transfected cells.

BACKGROUND OF THE INVENTION

The hormone erythropoietin plays a major role in regulatingerythropoiesis, the formation of red blood cells, and deficiencies oferythropoietin result in anemia. Detailed studies of the hormone andattempts at replacement therapy have been difficult due to the scarcityof purified material.

Normal production of human red blood cells requires the secretion oferythropoietin by the kidney, apparently as the mature glycoprotein. Inthe steady state this hormone circulates in the blood at a concentrationof 10 to 18 milliunits (128-230 picograms) per milliliter, and with thestimulus of severe tissue hypoxia (oxygen deficiency) the levels mayincrease as much as 1000-fold. The elevated hormone level triggersproliferation and differentiation of a population of receptive stemcells in the bone marrow, stimulates hemoglobin synthesis in maturingerythroid cells, and accelerates release of red cells from the marrowinto circulation, thereby increasing the red cell mass and amelioratingthe hypoxic conditions. Patients with deficiencies of erythropoietin,such as those with chronic renal failure, often suffer severe anemia.

Erythropoietin is a glycoprotein of 34-38 kd with approximately 40% ofits molecular weight provided by carbohydrate. At least one disulfidebridge is required for activity. Little is known about the structure ofthis hormone, and the details of its synthesis are not well understood.Recent isolations of cDNA and genomic clones provide opportunities toanalyze control of erythropoietin production, but the expression ofbiologically active human erythropoietin in sufficient quantities forreplacement therapy has not been achieved.

SUMMARY OF THE INVENTION

Pursuant to this disclosure, biologically active human erythropoietincan be expressed at high levels (at nominal titers exceeding two millionUnits per liter of supernatant) from stably transfected mammalian celllines. Thus, an abundant source of purified human erythropoietin forclinical applications is provided. Surprisingly high expression oferythropoietin is achieved by transfecting host cell lines with the ApaI restriction fragment of the human erythropoietin gene. The sensestrand of the Apa I restriction fragment has a nucleotide sequencecorresponding to that shown in FIG. 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B is a schematic representation of the subject 2426 bpApaI restriction fragment that contains the human erythropoietin genesequences;

FIG. 2 depicts a representative plasmid expression vector (pD11-Ep) thatcontains the 2426 bp Apa I restriction fragment; and,

FIG. 3 depicts another expression vector (pBD-EP) carrying the subjectApa I restriction fragment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the preferred embodiment, a genetically engineered construction ofthe ApaI restriction fragment shown in FIG. 1 is inserted into amammalian expression vector, such as those shown in FIGS. 2 and 3, andintroduced into mammalian cell lines to develop stably transfected cellsthat produce large amounts of biologically active human erythropoietin.The Apa I restriction fragment of the human erythropoietin gene wasselected to maximize efficient transcription of erythropoietin messengerRNA and effective translation and post-translational modification of theRNA into mature biologically active erythropoietin glycoprotein.Specifically, at the 5' end of the erythropoietin gene it was importantto remove interfering sequences but retain enhancing sequences. Intronswere retained in the Apa I restriction fragment in order to includepotentially significant enhancing sequences. At the 3' end of the genesome 3' untranslated sequences were retained to optimize putativeregulating sequences. The enhanced expression provided by the Apa Ifragment is demonstrated by the consistently high levels oferythropoietin expression described in the following working examples,using this fragment in concert with two promoters and two cell lines.

The following Examples are presented to illustrate the advantages of thepresent invention and to assist one of ordinary skill in making andusing the same. These Examples are not intended in any way to otherwiselimit the scope of the disclosure or the protection granted by LettersPatent hereon.

EXAMPLE 1

Isolation of Genomic Clones

A human genomic library in bacteriophage lambda (Cell 15:1157-1174,1978) was screened using low stringency hybridization conditions andmixtures of oligonucleotide probes as described in Cell 38:287-297,1984, hereby incorporated by reference.

Oligonucleotide mixtures were prepared using an Applied Biosystemssynthesizer and end-labeled using ³² p-ATP and T4 polynucleotide kinase.The synthetic oligonucleotides were designed to correspond to portionsof the amino terminal amino acid sequence of:

    H.sub.2 N-Ala-Pro-?-Arg-Leu-Ile-Leu-Asp-Ser-Arg-Val-Leu-Glu-Arg-Tyr-Leu-Leu-Glu-Ala-Lys-Glu-Ala-Glu-?-Ile-Thr-Asp-Gly-Gly-Ala                24

obtained by Yanagawa et al. (J.Biol.Chem. 259:2707-2710, 1984) for thehuman protein purified from urine of patients with aplastic anemia. Toreduce the degeneracy of the codons for the amino acid sequence of thisregion, the codon usage rules of Grantham et al. (Nucleic Acids Research8:43-59, 1981) and Jaye et al. (Nucleic Acids Research 11:2325-2335,1983) were employed. These rules take into account the relatively rareoccurrence of CpG dinucleotides in DNA of vertebrates and avoid, whereappropriate, potential A:G mismatch pairings. At amino acid position 24,an asparagine was placed as most likely (J.Biol.Chem. 259:2707-2710,1984). For the amino acids Glu-Ala-Lys-Glu-Ala-Glu-Asn, 2 pools of 72sequences each were synthesized to correspond to the predicted codons.Thus, one pool was TT(c/t)TC(a/g/t)GC(c/t)TC(c/t)TT(a/g/t)-GCTTC for the20 nucleotide probe, and the second pool replaced a T with a C atposition 18. For the amino acids Glu-Asn-Ile-Thr-Asp-Gly, one pool ofsequences (AGC TCC TCC ATC AGT ATT ATT T c/t!) was constructed for the23 nucleotide probe.

Plaques which hybridized to the oligonucleotide probes were rescreenedat lower density until pure. After initially positive phage clones wereplaque-purified, additional sequence information for the erythropoietingene was published by Jacobs et al. (Nature 313:806-310, 1985).Oligonucleotides were constructed from this information and used toconfirm the identity of the positive clones. After Eco RI restrictionenzyme digestion of the positive clones, insert DNA was gel-purified andligated by standard techniques into pUC 13 plasmid previouslyrestriction-digested with Eco RI. DNA sequence was determined bydideoxynucleotide chain termination (Proc.Natl.Acad.Sci.USA74:5463-5467, 1977) using dATP (α-³⁵ S) and the 17-mer universal primeror, in selected regions, specific oligonucleotide primers. ³² p-ATP wasfrom ICN; enzymes were from New England Biolabs or Bethesda ResearchLaboratories.

Approximately 4.8×10⁶ bacteriophage were screened by hybridization ofreplicate nitrocellulose filters. Three different clones remainedpositive through plaque purification, and the DNA insert wascharacterized by restriction mapping and partial dideoxynucleotidesequencing. Two of the three clones contained apparently completeinformation for the erythropoietin gene. The restriction map andsequence for the 2426 bp Apa I fragment of these clones is shown in FIG.1 and was essentially the same as that recently published by Jacobs etal. (Nature 313:806-810, 1985) for the gene for human erythropoietin.The low frequency of phage isolates containing the erythropoietin genein this amplified library, one in approximately 2×10⁶ bacteriophage, isconsistent with the suggestion that erythropoietin exists as a singlecopy in the human genome. Southern blot hybridization of total humangenomic DNA with the Apa I fragment or other restriction fragments ofthe erythropoietin gene indicated only a single hybridizing band with noadditional regions of highly homologous DNA.

EXAMPLE 2

Selection of the Apa I Restriction Fragment

For construction of expression vectors the Apa I restriction fragment ofthe erythropoietin gene was obtained using techniques described in Proc.Natl.Acad.Sci.USA 74:5463-5467, 1977, hereby incorporated by reference.Briefly, isolated phage DNA was digested with the restriction enzyme ApaI (Bethesda Research Laboratories) followed by separation on a 1%agarose gel and isolation by electroelution, phenol extraction andethanol precipitation. The fragment was confirmed by partial sequencingas in Example 1.

Referring to FIG. 1, the inserted Apa I restriction fragment contained58 bp of 5' untranslated sequences (nucleotides 0001-0058) followed bysequences coding for a putative 27 amino acid signal peptide, the matureprotein, four putative intervening sequences, and 222 bp of 3' noncodingDNA sequence. At the 5' end of the gene the Apa I site is located 58base pairs upstream from the ATG start codon (0058) for the proteinsequence. This 5' site (0001) was selected to avoid a false start sitejust upstream from the Apa I restriction site while including putativeregulatory sequences considered to be important for efficient ribosomalbinding and processing. The Apa I site at the 3' end (2426) was selectedto include putative processing signals in most of the 3' untranslatedsequences while removing further downstream sequences. The completeerythropoietin gene, including intron sequences, was used in order toinclude potential regulatory or enhancing sequences located in intronsthat might contribute to erythropoietin gene expression or proteinmodification and secretion.

EXAMPLE 3

Construction of Expression Plasmids Carrying the Apa I RestrictionFragment

The 2426 bp ApaI restriction fragment containing the intact humanerythropoietin gene was inserted into two expression vectors, the pD-11and pBD constructs, each of which is based on a different mammalianpromoter.

The plasmid expression vector pD11 was derived from a previouslydescribed plasmid (Nucleic Acids Research 13:841-857, 1985, expresslyincorporated herein by reference) and contained the simian virus 40(SV-40) enhancer sequences and origin of replication as well as theadenovirus-2 major late promoter and tripartite leader sequences. TheApa I fragment of human erythropoietin genomic sequences (FIG. 1) wasgel-purified, and single stranded ends were filled in by treatment withT4 DNA polymerase. Bam HI linkers were ligated to both blunt ends, andthe construct was inserted into a unique B-Bam HI restriction site ofpD11 in order to direct transcription of the erythropoietin gene from astrong promoter.

The structure of the resulting expression plasmid pD11-Ep carrying theApa I fragment (Ep) is depicted in FIG. 2. The plasmid pD11 contains 350bp of the adenovirus left-terminus (0-1), the origin and enhancersequences from SV-40 (E), the adenovirus major late promoter (MLP), theadenovirus-2 tripartite leader (L1-3) and third leader 5' splice site(5' ss), an immunoglobulin 3' splice site (3' ss), and the late SV-40polyadenylation signal (pA) in the Eco RI (RI) restriction site of pML.Recombinant plasmids were cloned in E. coli HB101 and purified byisopycnic centrifugation in cesium chloride. The expression plasmidpD11-Ep is approximately 6500 bp in length. The construction wasconfirmed by restriction mapping and partial dideoxynueleotidesequencing.

pBD contains the MT-I promoter sequence (Glanville, Durham & Palmiter,Nature 292:267-269, 1981, expressly incorporated herein by reference)and a DHFR selectable marker sequence carried in pUC plasmid. Referringto FIG. 3, the 2426 bp Apa I restriction fragment (EP) was inserted intoa unique Sma I restriction site in pBD to make expression plasmidpBD-EP. The DHFR sequence in pBD-EP was associated with origin andenhancer sequences from SV40 and with Hepatitis B Surface Antigenpolyadenylation sequences. pBD-EP was processed essentially as describedabove for pD11-EP.

While plasmid vectors were used in these confirming experiments, it iscontemplated that viral or retroviral expression vectors can likewise beused to introduce the Apa I erythropoietin gene fragment into host celllines. Suitable DNA viruses for this purpose would include adenovirus orBPV (Bovine Papilloma Virus). Suitable retroviral vectors are also knownand available. It is understood that such a retroviral (RNA) vectorwould carry the anti-sense strand of the Apa I restriction fragment,that is, one having a sequence corresponding to the RNA transcribed fromthe sense strand shown in FIG. 1 or to an allele thereof. The retroviralvector would also include sequences for trans-acting factors requiredfor reverse transcription of the viral genome and integration of the DNAform of the virus into the host genome.

EXAMPLE 4

Transfection of Mammalian Cells

Each of two mammalian cell lines was transfected with each of thepD11-EP and pBD-EP constructs. Mammalian cell lines, COS-7 (monkeykidney) and BHK (baby hamster kidney), were maintained in Dulbecco'smodified essential medium containing 10% fetal calf serum. Cells werepassaged and when 50-70% confluent were transfected by the calciumphosphate method (Virology 52:456-467, 1973). BHK derives from kidneyepithelial cells, generally regarded as the most likely cell thatproduces erythropoietin in the natural state when a mammal is anemic orhypoxic. Thus, these cells might recognize critical regulatory sequencesin the Apa I fragment of the erythropoietin gene, and then effectivelyprocess and produce the erythropoietin glycoprotein.

For transient expression of cells in a 100 mm culture dish a total of 20μg DNA was used: 10 μg of pD11-EP plasmid containing the erythropoietingene and 10 μg of carrier salmon sperm DNA. After 48 hours thesupernatant was collected, centrifuged at 400 g for 10 minutes to removecells and debris, and frozen at -20° C. The cells were harvestedseparately. The results of transfections with pD11-EP alone fortransient expression are shown in Table 1. Data are from 3 experimentsfor each cell type.

                  TABLE 1                                                         ______________________________________                                                   Erythropoietin per ml of culture                                                             Units                                               Mammalian cells                                                                            Micrograms protein                                                                         (in vitro bioassay)                                 ______________________________________                                        BHK          3.4 ± 0.2 270 ± 16                                         COS-7        3.2 ± 0.4 255 ± 32                                         ______________________________________                                    

The observed levels of erythropoietin secreted into the supernatant ofeither the COS-7 or BHK mammalian cell lines were approximately 80 timeshigher than those previously reported for transient expression of a cDNAcoding for erythropoietin or for transient expression of otherconstructs using intact erythropoietin genes.

To establish stable cell lines producing high levels of erythropoietin,either COS-7 or BHK cells were cotransfected with the pD11-Ep plasmidand pDHFR-1a, a plasmid containing a cDNA for dihydrofolate reductase ina similar mammalian expression vector. The transfection procedure wasmodified so that 5 μg of pD11-Ep plasmid, 5 μg of pDHFR-1a plasmid, and10 μg of carrier DNA were cotransfected. After additional incubation for18-24 hours, varying concentrations of methotrexate (10 nM to 1 mM) wereadded to the cultures. Cells that incorporated the DHFR gene would beviable in the selective medium. After incubation for several more days,viable colonies resistant to methotrexate were isolated, passaged andscreened for the presence of erythropoietin bioactivity in thesupernatant. Approximately half of the methotrexate-resistant coloniesthat were assayed secreted detectable erythropoietin activity.

To establish stable cell lines with the expression vector pBD-EP, BHKcells were transfected by the calcium phosphate method. After 18-24hours, these cultures were subjected to concentrations of methotrexatevarying from 1 μM up to 1 mM. After incubation for several more days,viable colonies resistant to these relatively high levels ofmethotrexate were isolated, passaged and screened. In this series, allof the methotrexate-resistant colonies that were assayed secreteddetectable erythropoietin activity.

In order to optimize the expression of the transcriptional unitcontaining the erythropoietin gene and the DHFR gene, BHK cell linescontaining either pBD-Ep or pD11-Ep and secreting high levels oferythropoietin were passaged several times into increasingconcentrations of methotrexate. However, rather than a gradual increasein the selective pressure on the cell lines by small incremental stepsin the concentration of methotrexate, the cells were immediatelychallenged with very high levels of methotrexate (i.e., 1 mM). Only afew cells survived, but those cells would have incorporated the plasmidconstruction into a region of DNA particularly advantageous forexpression (a so-called "hot spot") and/or would have many copies of theconstructed transcriptional unit. Thus, the highest producing cell lineswere selected in one step. Cell lines (including F 7.2 and S 5.2 listedin Table 2) were considered stable if erythropoietin production remainedhigh for more than 15 passages in the absence of methotrexate selectivepressure.

Amounts of erythropoietin activity in the cell pellets could not bedetermined due to the presence of significant inhibitors of the assay inthe cell extracts. Consequently, the results shown in Table 2 do notanalyze the intracellular levels of erythropoietin protein but ratherthe amount of erythropoietin protein produced and secreted into thesupernatant by the cell lines.

                  TABLE 2                                                         ______________________________________                                        Expression of Recombinant Erythropoietin                                      From Stably Transfected BHK Cell Lines                                                  Erythropoietin per ml of supernatant                                                         Units                                                Cell Line   Micrograms protein                                                                         (in vitro bioassay)                                  ______________________________________                                        pBD-EP                                                                        F 1.1       12.4          970                                                 F 3.4       32.0         2500                                                 F 6.1       79.6         6210                                                 F 7.2       84.1         6728                                                 pD11-EP                                                                       S 1.2        6.4          500                                                 S 2.4       64.2         5000                                                 S 5.2       82.1         6400                                                 ______________________________________                                    

The observed amounts of erythropoietin secretion correspond to nominalrates of up to almost seven million Units per liter. Such nominal ratesare determined by multiplying the observed yield per ml by one thousandto give an anticipated scaled-up production yield per liter.

The observed amounts of erythropoietin secretion using the Apa Ifragment were on the order of 300 times greater than those previouslyreported (Lin et al., Proc.Natl.Acad.Sci.USA 82:7580-7584, November1985) for a stably transformed CHO cell line containing a differentgenomic fragment of the human erythropoietin gene.

Control experiments for these transfection assays included supernatantsfrom nontransfected cells and parallel cultures of cells transfectedwith plasmids containing DNA encoding other proteins, includingbacterial chloramphenicol acetyl transferase and human coagulationprotein, factor IX. None of the control cultures, mock transfections, orcultured cells transfected with other genes had detectableerythropoietin activity. It was also noted in the above series ofexperiments with the erythropoietin gene that the levels of expressionobtained for selected cell lines were not related to whether theselectable marker was cotransfected along with the erythropoietin geneor was inserted into the Apa I fragment-containing plasmid prior totransfection (data not shown).

Other representative transfection methods suitable for practicing thisinvention include DEAE-dextran mediated transfection techniques,lysozyme fusion or erythrocyte fusion, scraping, direct uptake, osmoticor sucrose shock, direct microinjection, indirect microinjection such asvia erythrocyte-mediated techniques, and/or by subjecting the host cellsto electric currents. By transfection is meant the transfer of geneticinformation, and specifically the information encoded by the Apa Irestriction fragment of a human erythropoietin gene, to a cell usingisolated DNA, RNA, or synthetic nucleotide polymer. The above list oftransfection techniques is not considered to be exhaustive, as otherprocedures for introducing genetic information into cells will no doubtbe developed. The Apa I restriction fragment will typically be operablylinked (ligated) to other nucleic acid sequences such as promoter,enhancer, and polyadenylation sequences, prior to transfection. Whilehost cell lines of mammalian origin are described, and kidney epithelialcells are considered to be particularly preferred, it is contemplatedthat other eukaryotic as well as prokaryotic (bacteria or yeast) hostcell lines can be employed in the practice of this invention; veryrecent introductions of mammalian genes into plant cells offer thepotential for employing plant or algal cells as well.

EXAMPLE 5

Erythropoietin Expression From Transfected Cell Lines

The recombinant erythropoietin protein secreted into the supernatant ofthe transfected cell lines was biologically active, and large amounts ofthe hormone were secreted: up to 7000 units per milliliter.

The in vitro assay for erythropoietin biological activity was based onthe formation of erythroid colonies (from CFU-E; erythroidcolony-forming cells) in cultures of mouse bone marrow cells in plasmaclot (Blood Cells 4:89-103, 1978). The sensitivity of this assay isabout 5 milliunits/ml. The erythropoietin used as the standard for assaywas a partially purified preparation from plasma from anemic sheep(Connaught, Step 3 Ep, Lot 3026). Supernatants were assayed frompassaged cell lines grown for 24 hours in fresh medium withoutmethotrexate. The supernatant was diluted 1:200 with medium, and amountsbetween 1 and 10 microliters were added per milliliter of assay culturecontaining 2×10⁵ marrow cells, 10% bovine citrated plasma, 20% fetalcalf serum, 1% bovine serum albumin, and 1.6% beef embryo extract(Gibco). After incubation for 36 to 48 hours the plasma clots were fixedon microscope slides, stained with benzidine for hemoglobin, anderythroid colonies were enumerated. In the absence of addederythropoietin, no CFU-E-derived colonies were detected. Optimalerythroid colony growth (100-150 CFU-E detected per 2×10⁴ marrow cells)was observed routinely with 50 mU (0.64 nanograms) erythropoietin per mlof culture. Large amounts of erythropoietin hormones were secreted intothe supernatant of the transfected cell lines, see Tables 1 and 2, up to7000 units per milliliter. Assuming the recombinant erythropoietin has aspecific activity equivalent to that of natural erythropoietin (78,000units per mg protein), the biological assay corresponds to approximately80 μg of erythropoietin protein per milliter.

In addition, supernatants from selected cell lines were assayed forimmunologically reactive erythropoietin by competitive radioimmunoassayusing a polyvalent anti-human-erythropoietin rabbit anti-serum(J.Cell.Physiol. 118:87-96, 1984). The amount of protein measured by theradioimmunoassay was equivalent to the protein level estimated by thebiological assay. These data indicate that the transfected cell linesexpressed and secreted erythropoietin protein that was greater than 98%active.

The recombinant erythropoietin produced by the transfected cells wasfurther characterized to demonstrate that these cells were secretingauthentic hormone. Selected cell lines were assayed for in vivoerythropoietin activity in exhypoxic polycythemic mice (Nature191:1069-1087, 1961). Supernatants secreted from the cell lines hadpotent in vivo biological activity when assayed in the exhypoxicpolycythemic mouse. In experiments using partially purified nativeerythropoietin, it had been noted previously that neuraminidasetreatment completely abrogated erythropoietin activity when assayed inthe intact animal (J.Biol.Chem. 247:5159-5160, 1958). The loss ofactivity presumably was due to increased clearance by the liver of thedesialated hormone since neuraminidase-treated erythropoietin remainedfully active in vitro. The observation of potent in vivo biologicalactivity indicates that the transfected mammalian cell linesappropriately add carbohydrate and the terminal sialic acids to theerythropoietin protein during post-translational modification.

In separate experiments, the activity of erythropoietin in the in vitrobiological assay was neutralized by a neutralizing anti-humanerythropoietin antibody added to the culture.

The erythropoietin secreted into the supernatant of representativetransfected cell lines was also assayed for proliferative effects onother marrow progenitor cells. Recombinant erythropoietin was assayedfor its effect on a variety of progenitors from mouse and human marrowincluding erythroid colony-forming cells (CFU-E), erythroidburst-forming cells (BFU-E), granulocyte-macrophage precursors (CFU-GM),and mixed-cell colony-forming cells (CFU-Mix) (J.Cell.Physiol.Suppl.1:79-85, 1982; J.Cell.Physiol. 118:87-96, 1984). Erythroid stem cellsexhibited a proliferation response to the recombinant erythropoietinthat was parallel to the dose-response relationship found with naturalerythropoietin. Neither CFU-GM nor CFU-Mix exhibited any proliferativeresponse to the recombinant erythropoietin at concentrations up to 10units per milliliter of assay cell culture.

When analyzed by SDS-PAGE under reducing or nonreducing conditions thepurified recombinant erythropoietin electrophoresed identically toerythropoietin purified from urine of patients with aplastic anemia.Similar microheterogeneity of the proteins was observed, and thepredominant species had identical molecular weights of 34 kD.

While the present invention has been described in conjunction withpreferred embodiments, one of ordinary skill after reading the foregoingspecification will be able to effect various changes, substitutions ofequivalents, and other alterations to the compositions and methods setforth herein. It is therefore intended that the protection granted byLetters Patent hereon be limited only by the definition contained in theappended claims and equivalents thereof.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A recombinant DNAconsisting essentially of the 2.4 kb ApaI restriction fragment ofgenomic human erythropoietin gene.
 2. The recombinant DNA of claim 1having the nucleotide sequence depicted in FIGS. 1A and 1B.
 3. Arecombinant DNA construct comprising an insert consisting essentially ofthe 2.4 kb ApaI restriction fragment of genomic human erythropoietingene.
 4. A eukaryotic host cell transformed with a recombinant DNAconstruct comprising an insert consisting essentially of the 2.4 kb ApaIrestriction fragment of genomic human erythropoietin gene.
 5. Theeukaryotic host cell of claim 4 which is mammalian.
 6. The eukaryoticmammalian host cell of claim 5 which is baby hamster kidney.